CN104711225B - The external preparation method of NK cells - Google Patents
The external preparation method of NK cells Download PDFInfo
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Abstract
The present invention relates to a kind of NK cell preparation methods suitable for immunization therapy.A kind of external preparation method of NK cells is specifically provided, the NK cells are differentiated to form by embryo stem cell external evoked, and the preparation method includes the following aspects：The amplification cultivation of embryonic stem cell, induced differentiation of embryonic stem cells are candidate stem cell, CD34+Purifying, the amplification of hematopoietic cell, hematopoietic cell are induced to differentiate into NK cells.The present invention also provides Fiber differentiation systems prepared by NK cells in vitro.The preparation method of the present invention is simple, and the NK cell tumour target cell fragmentation effects obtained are notable.
Description
Technical field
The present invention relates to biological technical fields, are related to a kind of external preparation method of NK cells, specifically, are related to one kind
The method of NK cells is prepared into from induced differentiation of embryonic stem cells.
Background technology
NK cells（Natural killer cells) natural killer cells is also known as, it is a kind of CD56+, CD3-Lymph it is thin
Born of the same parents are the effector cells of natural immune system in body, it is recognizable tumour and virus infection without antigen presensitization
Cell.It is located at the first line of defence of body defenses system, while it can pass through Early insulin secretion cytokine profiles and chemotactic again
The factor adjusts Acquired immune response, therefore is also the bridge of the connection innate immunity and acquired immunity, in tumour immunity, removes
Non- own cell etc. plays a significant role.
It is the method using in vitro culture clinically to obtain NK cell therapies at this stage, the NK in patient itself peripheral blood
Cell is expanded, and makes cell number expansion thousands of times and cytotoxic activity greatly enhances, then feed back the internal of patient.It is derived from trouble
The cell of person itself limits the possibility of its industrialization production first；It is low additionally, due to the content of NK cells in peripheral blood, only
The 5%-10% of total number of peripheral blood is accounted for, cell quantity is limited, and still lacks the amplification in vitro of effective high-purity at present
System, so greatly limiting the clinical practice of NK cells, this traditional NK cell preparation methods are not reason
The selection thought.First, it is mainly used for man-to-man individualized treatment method, can only carry out in hospital's bedside；Second, it is this to control
The time for the treatment of method natural killer cell induction is too long, at least needs 6 weeks, for patients with advanced cancer, especially after operation
Radiotherapy, the patient of chemotherapy, may be just dead before cell prepares completion or cell is fed back；3rd, man-to-man treatment,
I.e. everyone will do the process of an a full set of cell induced amplification culture, and in terms of laboratory room managing, indoor quality control is difficult,
Between ward quality monitoring is more difficult to；4th, the NK cells of many patients in itself are exactly defect, and adoptive therapy effect is limited.
Although there is substantial amounts of research to concentrate on the CD34 for finding separate sources at present+Cell breaks up most to the induction of NK cells
Good system, but there has been no a kind of methods can simply, efficiently obtain the strong NK cells of high-purity, high quantity, killing ability.
With the development and progress of scientific research, it is induced to differentiate into NK cells using stem cell and is possibly realized, be a great suction
The selection of gravitation, has broad application prospects.Embryonic stem cell is exactly outstanding person therein, and embryonic stem cell has height
Undifferentiated and very strong multiplication capacity can be divided into the various cells of tissue.At present, human embryo stem cell by
Successfully it is divided into each germinal layer of ripe and function subset, the hemopoietic system including cell.
The content of the invention
The purpose of the present invention is being directed to problems of the prior art and deficiency, one kind is provided and is induced by embryonic stem cell
The external preparation method of NK cells is cultivated, industrialization is limited and be difficult to solve NK cell deriveds deficiency, curative effect in the prior art
The problem of production, has broad application prospects.
Invention also provides the Fiber differentiation systems of the external preparation of NK cells, simplify purifying and identification of cell product
Program, avoid virus pollute caused by harm.
To achieve the above object, the present invention adopts the technical scheme that：A kind of external preparation method of NK cells, it is specific to wrap
Include following steps：
（1）The in vitro culture of embryonic stem cell
Using the embryonic stem cell culture system of no feeder layer, in the pretreated culture of gelatin after embryonic stem cell recovery
Adhere-wall culture in ware, after cultivating 2-3d, digestive juice had digestive transfer culture.
（2）Inducing embryo stem cell is to hematopoetic cell differentiation
The method being combined using stroma cell co-cultivation with cytokine induction, embryonic stem cell is broken up with 2mL to be cultivated
After liquid is resuspended, it is inoculated in and is covered in advance in 6 orifice plates of OP9 cells, be put into 37 DEG C, 5%CO2, 95% saturated humidity culture
Case culture, and add cell factor Flt3 ligands（FL）, interleukin-15（IL-15）, interleukin-6（IL-6）, in vain
Cytokine -7 (IL-7), Kit ligands（KL）.Culture 1 day, embryonic stem cell forms the cell colony to suspend, these cells
Colony is transferred in ultralow absorption culture dish, and adds above-mentioned cell factor, and cell is collected after 2-3 days.
（3）Immuno magnetic cell separation purifies CD34+Cell
Since candidate stem cell is distributed mainly on CD34+In cell, therefore CD34 is purified using immunomagnetic beads method+Hematopoietic Stem
Cell, and with the separated obtained CD34 of flow cytomery+Cell purity.
（4）CD34+The expansion culture of cell
By CD34 after purification+Cell is with 1.0 × 104Cells/mL density inoculation serum free medium in, be placed in 37 DEG C,
5%CO2, 95% saturated humidity incubator in quiescent culture, every 7 days half amounts change liquid.Equal factor-containing in culture solution：Stem cell because
Sub (SCF), interleukin 3 (IL-3), thrombopoietin (TPO), erythropoietin(EPO) (EPO) and interleukin-6
(IL-6).Culture collects cell in 2-3 weeks.
（5）CD34+Cell induction differentiation NK cells
CD34+It when cell is expanded to sufficient amount, is resuspended with serum free medium, and adds combination of cytokines 1.：It is dry
Cell factor (SCF), human granulocyte macrophage colony stimulus factor (GM-CSF) and Flt3 ligands（FL）, 37 DEG C, 5%CO2With
The incubator culture of 95% humidity, 24 it is small when after, cell is transferred to new Tissue Culture Flask and continues to cultivate, add cell factor
Combination is 2.：Interleukin 2 (IL-2), interleukin 12 (IL-12), interleukin-15 (IL-15), monoclonal resist
Body anti-CD3 (anti-CD3), interleukin 4 (IL-4) and interleukin 7 (IL-7), are positioned over 37 DEG C, 5%CO2、
The incubator culture induced NK cell of 95% saturated humidity, changes the liquid once every three days, and supplements combination of cytokines 2., induces 4 weeks
Flow cytometer detection CD3 afterwards-CD56+NK cells.
（6）The measure of NK cell killing activities.
Further, the step（1）It is trained in the Process of in vitro of embryonic stem cell using the DMEM of no feeder layer
The system of supporting, the culture solution composition of the embryonic stem cell are：DMEM culture mediums, addition 20%KnockOut serum substitutes,
0.1mM 2 mercapto ethanols, 100 μ g/mL L-Glutamines, 1% nonessential amino acid, the Leukocyte factor of 1000U/mL
LIF, 0.1mM MEM.
Further, the step（2）Inducing embryo stem cell is trained altogether into hematopoetic cell differentiation using stroma cell
The method being combined with cytokine induction is supported, ES cell differentiation culture solution used composition is：α-MEM culture mediums, body
Fraction 10%KnockOut serum replacements, 100 μm of ol/L thioglycerols (MTG)；Inducible factor used is combined as：5ng/
mL FL + 20ng/mL IL-15 + 20ng/mL IL-6 + 10 ng/mL IL-7 + 10ng/mL KL.The present invention uses
The culture medium of serum-free candidate stem cell avoids the pollution of heterologous gene.
Further, the step（4）To obtain enough pure CD34+Cell, using first immunomagnetic beads point
From purifying CD34+Cell, it is rear to expand culture CD34+The method of cell, and serum free medium is used in incubation：IMDM
Culture medium, 5 × 10-5M beta -mercaptoethanols, 1%BSA, 0.01mg/mL insulin, 0.7mg/mL transferrins, 2 × 10-6M courages are consolidated
Alcohol, 20mmol/100mL L-Glutamines；Combinations of factors used is 50ng/mL SCF+20ng/mL in amplification cultivation
IL-3 + 4U/mL TPO + 3U/mL EPO + 20ng/mL IL-6。
Further, the step（5）CD34+It is repeatedly induced using cell factor in cell induction differentiation NK cells
Method obtains enough NK cells, 1. combination of cytokines used is： 30ng/mL SCF + 30ng/mL GM-CSF +
5ng/mL FL；2. combination of cytokines used is：10ng/mL IL-2 + 20ng/mL IL-12 + 20ng/mL IL-15
+ 10ng/mL anti-CD3 + 20ng IL-4 + 10ng/mL IL-7。
The preparation method of the present invention possesses advantages below：
（1）It solves the problems, such as that NK cell deriveds are insufficient, new thinking is provided for NK cell therapies.
（2）The program of purifying and identification of cell product is simplified using free serum culture, caused by avoiding virus pollution
Harm.
（3）In the embryo stem cell for directional hematopoetic cell differentiation stage, initiate and be with the addition of inducible factor 5ng/mL FL, 20ng/
ML IL-15,20ng/mL IL-6,10 ng/mL IL-7,10ng/mL KL, significantly shorten the Fiber differentiation time, improve
The motility rate of hematopoietic cell.
（4）Chain induction has creatively been carried out for NK cell stages in hematopoietic cell directed differentiation, has been added in inoculation step
Inducible factor combination is added 1.：30ng/mL SCF, 30ng/mL GM-CSF, 5ng/mL FL, after cultivating two days, addition induction
Combinations of factors is 2.：10ng/mL IL-2、20ng/mL IL -12、20ng/mL IL-15、10ng/mL anti-CD3、20ng
IL-4、10ng/mL IL-7.The NK cytoactives obtained by this method are high, can reach more than 97%, and the tumour of NK cells
Fragmentation effect is notable.
Description of the drawings
Attached drawing described herein is used for providing a further understanding of the present invention, forms the part of the application, this hair
Bright schematic description and description does not constitute improper limitations of the present invention for explaining the present invention.In the accompanying drawings：
Fig. 1 is the technical solution implementing procedure figure of the external preparation method of NK cells.
Specific implementation method
It is below with reference to the accompanying drawings and in conjunction with the embodiments, next that the present invention will be described in detail.With reference to Fig. 1：The external preparation of NK cells
Shown in the technical solution implementing procedure figure of method, the specific embodiment of the invention is as follows：
The in vitro culture of 1 embryonic stem cell of embodiment：
1. by the embryonic stem cell preserved in liquid nitrogen and 37 DEG C of water-baths, flash melt, cell suspension becomes liquid from solid-state.
2. above-mentioned cell suspension inoculation is positioned over 37 DEG C, 5%CO in the pretreated culture dish of gelatin2, 95% saturation
The incubator culture of humidity cultivates 2-3d, after cell fusion degree reaches 85%-90%, with digestive juice had digestive transfer culture.
3. according to 1:3-1:5 passages supply culture solution and continue to cultivate into new culture dish.
4. per 2-3d digestive juice had digestive transfer cultures.
2 inducing embryo stem cell of embodiment breaks up to candidate stem cell
1. the OP9 cells that will be frozen using first 1 day, with 1.5 × 105Cells/mL cell concentrations be inoculated into it is pre- be covered with it is bright
In 6 well culture plates of glue.
2. the 2nd day centrifuges embryonic stem cell, clear liquid is abandoned, after being resuspended with differentiation culture solution, be inoculated in has OP9 in advance
In 6 orifice plates of cell, 37 DEG C are put into, 5%CO2, 95% saturated humidity incubator culture, and add cell factor 5ng/mL FL,
20ng/mL IL-15,20ng/mL IL-6,10 ng/mL IL-7,10ng/mL KL.
3. the ES broken up is transferred in new ultralow absorption culture dish by culture 1d, and adds above-mentioned cell factor.
4. after culture 2-3 days, cell is collected by centrifugation, PBS liquid is added to clean cell, centrifugation（1500r/min, 10min）After go
Supernatant, then add PBS liquid that cell, eccentric cleaning 3 times is resuspended；After adding in a small amount of culture solution to cell after cleaning, cell is made and hangs
Liquid counts, spare.
3 immuno magnetic cell separation of embodiment purifies CD34+Cell
1. by the cell of collection with 1 × 108A monocyte is suspended from 300 μ l PBS buffer solution, add in 100 μ l Fc by
Body blocking agent, then add in 100 μ l AntiCD3 McAbs, 4 immunomagnetic beads thereto, mixing, 4 DEG C of incubation 30min, then with PBS buffer solution from
The heart washs 2 times, centrifuges 10min every time with the speed of 1000rpm, and the 500 μ l PBS buffer solution washed cell that suspends again is close
It spends for 108A/500 μ l.
2. splitter is fixed in MACS magnetic fields, mark cell is slow transitted through into MiniMACS splitters, elution removal
CD34-Splitter is removed magnetic field by cell, is pressurizeed with 1mL buffer solutions and is eluted splitter, collects CD34+Cell simultaneously counts.With stream
Formula Cytometric Analysis CD34+Cell purity.The result shows that the CD34+The purity of candidate stem cell is more than 96%.
4 CD34 of embodiment+The expansion culture of cell
It will be after purification rich in CD34+Cell is with 1.0 × 104Cells/mL density is inoculated in 24 porocyte culture plates,
In 37 DEG C, 5%CO2, 95% saturated humidity incubator in quiescent culture, every 7 days half amounts change liquid.Using serum free medium and add
Add 50ng/mL SCF, 20ng/mL IL-3,4U/mL TPO, 3U/mL EPO and 20ng/mL IL-6, and add dual anti-（Mould
Plain 100IU/L streptomysins 100mg/L）.Culture collects cell in 2-3 weeks.
Free serum culture based component used：IMDM culture mediums, 5 × 10-5M beta -mercaptoethanols, 1%BSA, 0.01mg/mL pancreas
Island element, 0.7mg/mL transferrins, 2 × 10-6M cholesterol, 20mmol/100mL L-Glutamines.
5 CD34 of embodiment+Cell induction differentiation NK cells
1. cell inoculation：CD34+When cell is expanded to sufficient amount, be resuspended with serum free medium, and add cell because
Son：30ng/mL SCF, 30ng/mL GM-CSF, 5ng/mL FL, 37 DEG C, 5%CO2, 95% saturated humidity incubator culture.
2. transfer culture：It after cell culture 2 days after inoculation, is transferred to Tissue Culture Flask and continues to cultivate, add in serum-free
Culture medium, and add the factor：10ng/mL IL-2、20ng/mL IL -12、20ng/mL IL-15、10ng/mL anti-CD3、
20ng IL-4,10ng/mL IL-7 are positioned over 37 DEG C, 5%CO2, 95% saturated humidity incubator culture induced NK cell 5 days.
3. expand culture：It changes the liquid once every three days, and the factor that supplements the nutrients.Flow cytometer detection CD3 after inducing 4 weeks-CD56+NK
Cell.The result shows that the CD3-CD56+The purity of NK cells is more than 97%.
The measure of 6 NK cell killing activities of embodiment
Measuring method is discharged using 4h LDH, detection NK cells are to the fragmentation effect of Leukemia K562 cell.
1. passage cell strain K562 cells is taken to be counted, 1 × 10 is made5Cells/mL cell suspensions add in 96 orifice plates,
Per 50 μ l of hole.
2. by the NK cells of culture respectively according to effect：Target is 1:1、10:1、20:1、40:1 adds in 96 orifice plates, sets simultaneously
Effector cell and target cell Spontaneous release hole, culture medium Spontaneous release hole, target cell maximum release aperture, volume correction control, often
100 μ l of pore volume, are all provided with 3 multiple holes, and 250g centrifugation 4min are placed in 37 DEG C, 5%CO2, 95% saturated humidity incubator in be incubated
4h。
3. 45min target cells maximum release aperture adds in 10 μ l lysates per hole before reaction terminates.After reaction, per hole
It draws 50 μ l supernatants, 50 μ l LDH enzyme reaction solutions and is placed in another 96 new orifice plates, room temperature is protected from light 30min, adds in reaction eventually
Only 50 μ l of liquid, microplate reader survey its OD value.
4. calculate Nk Cell Activity.Formula is：Nk Cell Activity %=（Measure pipe OD values-target cell Spontaneous release pipe
OD values-effector cell's Spontaneous release pipe OD values）/（The maximum release pipe OD values-target cell Spontaneous release pipe OD values of target cell）×
100%。
Table 1：Difference effect targets than cell killing rate compare
* the P ＜ 0.05 compared with 1: 1.
Experimental result：As shown in table 1, each effect target prepared by the method for the present invention, which compares, can kill target cell, and with
Effect target than rise NK cells the killing activity of target cell is increased, in effect target ratio for 40:It can reach more than 90% when 1.
The foregoing is only a preferred embodiment of the present invention, is not intended to limit the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.Within the spirit and principles of the invention, that is made any repaiies
Change, equivalent substitution, improvement etc., should all be included in the protection scope of the present invention.
Claims (6)
1. a kind of external preparation method of NK cells, which is characterized in that comprise the following steps：
Step 1）The in vitro culture of embryonic stem cell：Using the embryonic stem cell culture system of no feeder layer, embryonic stem cell is multiple
After Soviet Union in the pretreated culture dish of gelatin adhere-wall culture, cultivate 2-3d after, digestive juice had digestive transfer culture；
Step 2）Inducing embryo stem cell is to hematopoetic cell differentiation：After embryonic stem cell is resuspended with 2mL differentiation culture solutions, it is inoculated in
It is covered in advance in 6 orifice plates of OP9 cells, is put into 37 DEG C, 5%CO2, 95% saturated humidity incubator culture, and add thin
Intracellular cytokine Flt3 ligands（FL）, interleukin-15（IL-15）, interleukin-6（IL-6）, interleukin 7
（IL-7）, Kit ligands（KL）；Culture 1 day, embryonic stem cell form the cell colony to suspend, these cell colonies are transferred to
In ultralow absorption culture dish, and above-mentioned cell factor is added, cell is collected after 2-3 days；
Step 3）Immuno magnetic cell separation purifies CD34+Cell：CD34 is purified using immunomagnetic beads method+Cell, and use flow cytometer
Detect separated obtained CD34+The purity of cell；
Step 4）CD34+The expansion culture of cell：By CD34 after purification+Cell is with 1.0 × 104Cells/mL density is inoculated with nothing
In blood serum medium, 37 DEG C, 5%CO are placed in2, 95% saturated humidity incubator in quiescent culture, every 7 days half amounts change liquid；Culture solution
In equal factor-containing：Stem cell factor（SCF）, interleukin 3（IL-3）, thrombopoietin（TPO）, red blood cell life
Cheng Su（EPO）And interleukin-6（IL-6）；Culture collects cell in 2-3 weeks；
Step 5）CD34+Cell induction differentiation NK cells：CD34+When cell is expanded to sufficient amount, with free serum culture base weight
It is outstanding, and add combination of cytokines 1.：Stem cell factor（SCF）, human granulocyte macrophage colony stimulus factor（GM-CSF）
With Flt3 ligands（FL）, 37 DEG C, 5%CO2, 95% saturated humidity incubator culture, 24 it is small when after, cell is transferred to new thin
Born of the same parents' blake bottle continues to cultivate, and addition combination of cytokines is 2.：Interleukin 2（IL-2）, interleukin 12（IL-12）、
Interleukin-15（IL-15）, monoclonal antibody anti-CD3（anti-CD3）, interleukin 4（IL-4）And leucocyte
Interleukin -7（IL-7）, it is positioned over 37 DEG C, 5%CO2, 95% saturated humidity incubator culture induced NK cell, change liquid one every three days
It is secondary, and supplement combination of cytokines 2., flow cytometer detection CD3 after inducing 4 weeks-CD56+NK cells；
Step 6）The measure of NK cell killing activities.
2. the external preparation method of NK cells according to claim 1, which is characterized in that the NK cells are by embryo
Stem cell Induction of committed differentiation, and being prepared using serum-free Fiber differentiation system in vitro.
3. the external preparation method of NK cells according to claim 1, which is characterized in that the step 1）Embryo does carefully
Using the DMEM cultivating systems of no feeder layer, the cultivating system of the embryonic stem cell forms is the in vitro culture of born of the same parents：DMEM
Culture medium adds in 20%KnockOut serum substitutes, 0.1mM 2 mercapto ethanols, 100 μ g/mL L-Glutamines, 1%
Leukocyte factor LIF, 0.1mM MEM of nonessential amino acid, 1000U/mL.
4. the external preparation method of NK cells according to claim 1, which is characterized in that the step 2）Middle induction embryo
Tire stem cell forms to the ES cell differentiation cultivating system used in hematopoetic cell differentiation：α-MEM culture mediums, volume integral
Several 10% KnockOut serum replacements, 100 μm of ol/L thioglycerols（MTG）；Inducible factor used is combined as：5ng/mL
FL+20ng/mL IL-15+20ng/mL IL-6 +10ng/mL IL-7+10ng/mL KL。
5. the external preparation method of NK cells according to claim 1, which is characterized in that the step 4）Middle CD34+Carefully
The expansion serum free culture system that uses of culture of born of the same parents for：IMDM culture mediums, 5 × 10-5M beta -mercaptoethanols, 1%BSA,
0.01mg/mL insulin, 0.7mg/mL transferrins, 2 × 10-6M cholesterol, 20mmol/100mL L-Glutamines；Amplification training
Combinations of factors used is 50ng/mL SCF+20ng/m L IL-3+4U/mL TPO+3U/mL EPO+20ng/mL in supporting
IL-6。
6. the external preparation method of NK cells according to claim 1, which is characterized in that the step 5）Middle CD34+Carefully
Born of the same parents induce differentiation NK cells to use step 4）Serum free culture system, 1. the combination of cytokines of addition is specially：30ng/mL
SCF +30ng/mL GM-CSF+5ng/mL FL；2. combination of cytokines is specially：10ng/mL IL-2+20ng/mL IL-12
+20ng/mL IL-15+10ng/mL anti-CD3 +20ng/mL IL-4+10ng/mL IL-7。
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| CN105441390A (en) * | 2015-11-18 | 2016-03-30 | 深圳爱生再生医学科技有限公司 | In-vitro three-dimensional amplification culture method for NK cells |
| CN107779433A (en) * | 2016-08-30 | 2018-03-09 | 天津市康婷生物工程有限公司 | Easily stimulate NK cells propagation and the feeder layer preparation method of differentiation |
| CN106434556B (en) | 2016-11-22 | 2019-10-11 | 上海新长安生物科技有限公司 | A kind of method of external evoked amplification I type NKT cell |
| CN107418931A (en) * | 2017-08-17 | 2017-12-01 | 重庆斯德姆生物技术有限公司 | A kind of cell culture fluid for improving motility rate |
| CN114507641B (en) * | 2022-04-20 | 2022-07-12 | 山东兴瑞生物科技有限公司 | Method for inducing and differentiating human embryonic stem cells into NK cells |
| CN115896019B (en) * | 2023-02-23 | 2023-05-26 | 山东兴瑞生物科技有限公司 | Method for inducing and differentiating induced pluripotent stem cells into NK cells |
| CN117305241B (en) * | 2023-11-28 | 2024-03-19 | 上海兴瑞一达生物科技有限公司 | Method for inducing and differentiating hiPSCs into NK cells |
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